Cytokines represent a diverse group of regulatory proteins with numerous biological functions including cell differentiation, cell growth, and cytotoxity. Inflammatory cytokines such as Tumor Necrosis Factor alpha and (TNF-.alpha.)and IL-1 (interleukin-1) have been shown to play pivotal roles in immune and inflammatory responses (McIntyre, T. M., et al., Thromb. Haemos. 1997, 78, 302-305). One of the most important effector functions of these cytokines is their ability to induce profound changes in the vascular endothelium (Introna, M. and Mantovani, A., Art. Thromb. and Vasc. Biol. 1997, 17, 423-428). Central to the process of inflammation is the induction of cell adhesion molecules on the endothelial cell surface, contributing significantly to the adherence and recruitment of circulating leukocytes to inflammatory sites. Upon exposure to TNF-.alpha. or IL-1, which are produced in response to injury or infection, cytokine receptors on endothelial cells activate a variety of intracellular signaling molecules. These signaling events result in the activation of specific transcription factors such as NF-kB and upregulate the expression of E-selectin, ICAM-1, VCAM-1, and other cell adhesion molecules (McIntyre, T. M., et al., Thromb. Haemos. 997, 78, 302-305; Introna, M. and Mantovani, A., Art. Thromb. and Vasc. Biol. 1997, 17, 423-428; Mantovani, A., et al., Thromb. Haemos. 1997, 78, 406-414). E-selectin has been shown to mediate the initial attachment and rolling of leukocytes along the vessel wall, whereas ICAM-1 and VCAM-1 are involved in the firm adhesion of leukocytes to the vessel wall and their transmigration through the vessel wall. E-selectin is rapidly and transiently induced by cytokines with peak expression occurring approximately 4-6 hours after exposure and returning to basal levels approximately 24 hours post exposure. In contrast, induction of ICAM-1 and VCAM-1 by cytokines is slower and persists for 24 hours or longer (Mantovani, A., et al., Thromb. Haemos. 1997, 78, 406-414; Dunon, D., et al., Curr. Opin. Cell Bio, 1996, 8, 714-723; Bischoff, J., Cell Adhes. and Angiog., 1997, 99, 373-376).
Responses to TNF-.alpha. are mediated through interactions with two distinct membrane receptors, termed TNFRI (TNF-.alpha. receptor I) and TNFRII (TNF-.alpha. receptor II). Two distinct families of adaptor proteins associated with TNF-.alpha. receptors have been identified. The death domain-containing proteins (e.g., TRADD) appear to couple the receptors to programmed cell death (Fiers, W., et al., J. Inflam., 1996, 47, 67-75; Wallach, D., et al., FEBS Lett., 1997, 410, 96-106; Hsu, H., et al., Cell, 1996, 84, 299-308), whereas the TRAF (TNF receptor associated factor) domain-containing proteins link the receptors to activation of specific transcription factors (Hsu, H., et al., Cell, 1996, 84, 299-308; Baeuerle, P. A., Curr. Biol., 1998, 8, R19-R22). Among the six members of the TRAF family that have been identified so far, TRAF2 has been reported to be important for TNF-.alpha.-mediated activation of JNK (c-Jun N-terminal kinase), as well as two major transcription factors, NF-kB (nuclear factor-kB) and AP-1 (activator protein 1) (Hsu, H., et al., Cell, 1996, 84, 299-308; Baeuerle, P. A., Curr. Biol., 1998, 8, R19-R22; Natoli, G., et al., J. Biol. Chem., 1997, 272, 26079-26082; Liu, Z. G., et al., Cell, 1996, 87, 565-576; Song, H. Y., et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 9792-9796). Both transcription factors play pivotal roles in the regulation of multiple genes including those involved in immune and inflammatory responses. AP-1 is activated by various MAPKs (mitogen-activated protein kinase) including ERK (extracellular-signal-regulated kinase), JNK and p38 MAPK (Fiers, W., et al., J. Inflam., 1996, 47, 67-75; Eder, J., TIPS, 1997, 18, 319-322). NF-kB is constitutively present in the cytosol of endothelial cells and kept inactive by association with inhibitory IkB family proteins. Upon exposure to TNF-.alpha., IKK (IkB kinase) phosphorylates IkB and initiates its ubiquitination and subsequent degradation. The released NF-kB translocates to the nucleus and participates in transcriptional activation (Collins, T., et al., FASEB J., 1995, 9, 899-909; Stancovski, I., and Baltimore, D., Cell, 1997, 91, 299-302).
Other signaling molecules, including MEKK1, pp90rsk (ribosomal S6 protein kinase), ras, and raf, have been implicated in the activation of NF-kB (Schulze-Osthoff, K., et al., Immunobiol., 1997, 198, 35-49). ras family members (Ha-ras, Ki-ras, N-ras) are GTP-binding proteins that act as major mediators in the regulation of cell proliferation and differentiation in response to a variety of extracellular stimuli including TNF-.alpha. (Bos, J. L., Biochem. Biophys. Acta, 1997, 1333, M19-M31). ras proteins have been shown to activate both the raf/MEK/ERK pathway as well as MEKK/JNKK/JNK pathway (Bos, J. L., Biochem. Biophys. Acta, 1997, 1333, M19-M31; Marais, R., and Marshall, C. J., Cancer Surveys, 1996, 27, 101-125; Adler, V., et al., J. Biol. Chem., 1996, 271, 23304-23309; Faris, M., et al., J. Biol. Chem., 1996, 271, 27366-27373; Terada, K., et al., J. Biol. Chem., 1997, 272, 4544-4548). raf family members (A-, B-, c-raf) are serine/threonine protein kinases that transmit signals from cell surface receptors to a variety of intracellular effectors including the MAPK pathways (Marais, R., and Marshall, C. J., Cancer Surveys, 1996, 27, 101-125; Daum, G., et al., TIBS, 1994, 19, 474-480). Besides ras, a variety of protein kinases including Src family kinases and PKC (protein kinase C) can potentiate raf activity (Marais, R., et al., J. Biol. Chem., 1997, 272, 4378-4383; Ueffing, M., et al., Oncogene, 1997, 15, 2921-2927). The major downstream effectors of raf are MEK/MKK1 (MAP kinase kinase 1) and MEK/MKK2 (MAP kinase kinase 2) which in turn phosphorylate and activate ERK1/2, and ultimately activate specific transcription factors (Marais, R., and Marshall, C. J., Cancer Surveys, 1996, 27, 101-125; Daum, G., et al., TIBS, 1994, 19, 474-480). Both ras and raf had been suggested to participate in the activation of NF-kB transcription factors (Schulze-Osthoff, K., et al., Immunobiol., 1997, 198, 35-49; Folgueira, L., et al., J. Virol., 1996, 70, 2332-2338; Koong, A. C., et al., Cancer Res., 1994, 54, 5273-5279; Bertrand, F., et al., J. Biol. Chem., 1995, 270, 24435-24441; Kanno, T., and Siebenlist, U., J. Immunol., 1996, 157, 5277-5283).
In many human diseases with an inflammatory component, the normal, homeostatic mechanisms which attenuate the inflammatory responses are defective, resulting in damage and destruction of normal tissue. For example, VCAM-1 may play a role in the metastasis of melanoma, and possibly other cancers. In addition, data have demonstrated that ICAM-1 is the cellular receptor for the major serotype of rhinovirus, which account for greater than 50% of common colds. (Staunton, et al., Cell, 1989, 56, 849-853; Greve et al., Cell, 1989, 56, 839-847).
Expression of ICAM-1 has also been associated with a variety of inflammatory skin disorders such as allergic contact dermatitis, fixed drug eruption, lichen planus, and psoriasis (Ho, et al., J. Am. Acad. Dermatol., 1990, 22, 64-68; Griffiths and Nickoloff, Am. J. Pathology, 1989, 135, 1045-1053; Lisby, et al., Br. J. Dermatol., 1989, 120, 479-484; Shiohara, et al., Arch. Dermatol., 1989, 125, 1371-1376). In addition, ICAM-1 expression has been detected in the synovium of patients with rheumatoid arthritis (Hale, et al., Arth. Rheum., 1989, 32, 22-30), pancreatic B-cells in diabetes (Campbell, et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 4282-4286), thyroid follicular cells in patients with Graves' disease (Weetman, et al., J. Endocrinol., 1989, 122, 185-191), and with renal and liver allograft rejection (Faull and Russ, Transplantation, 1989, 48, 226-230; Adams, et al., Lancet, 1989, 2, 1122-1125).
Inhibitors of ICAM-1, VCAM-1 and ELAM-1 expression would provide a novel therapeutic class of anti-inflammatory agents with activity towards a variety of inflammatory diseases or diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, various dermatological conditions, and psoriasis. In addition, inhibitors of ICAM-1, VCAM-1, and ELAM-1 may also be effective in the treatment of colds due to rhinovirus infection, AIDS, Kaposi's sarcoma and some cancers and their metastasis. The use of neutralizing monoclonal antibodies against ICAM-1 in animal models provide evidence that such inhibitors if identified would have therapeutic benefit for asthma (Wegner, et al., Science, 1990, 247, 456-459), renal allografts (Cosimi, et al., J. Immunol., 1990, 144, 4604-4612), and cardiac allografts (Isobe, et al., Science, 1992, 255, 1125-1127). The use of a soluble form of ICAM-1 molecule was also effective in preventing rhinovirus infection of cells in culture (Marlin, et al., Nature, 1990, 344, 70-72).
Current agents which affect intercellular adhesion molecules include synthetic peptides, monoclonal antibodies, soluble forms of the adhesion molecules, and antisense oligonucleotides. Antisense oligonucleotides to cell adhesion molecules are disclosed in U.S. Pat. Nos. 5,514,788 and 5,591,623, herein incorporated by reference. These have been directed against a single cell adhesion molecule. Additional agents are desired. Furthermore, a broader approach, targeting several adhesion molecules with a single agent may have several advantages, including economies of scale, broad spectrum utility, etc. Thus, an approach to target a molecule in the TNF-.alpha. signaling pathway may be a useful therapeutic treatment, providing a means to regulate multiple cell adhesion molecules with a single agent.
Inhibitors of molecules in TNF-.alpha. mediated signaling have been used to study the signal transduction pathways and suggest utility in the design of pharmacological agents. Inhibitors that have been used include DMSO (Essani, N. A., et al., Shock, 1997, 7, 90-96) against NF-kB, protein tyrosine kinase inhibitors (Adamson, P., et al., Cell Adhes. Commun.,1996, 3, 511-525; Pai, R., et al., J. Immunol., 1996, 156, 2571-2579), protein tyrosine kinase C inhibitors (Ballestas, M. E. and Benveniste, E. N., Glia, 1995, 14, 267-278), ubiquitin ligase inhibitors (Yaron, A., et al., EMBO J., 1997, 16, 6486-6494), and phospholipase A2 inhibitors (Thommesen, L., et al., J. Immunol., 1998, 161, 3421-3430). In addition, drugs that elevate cyclic AMP have been found to inhibit ELAM-1 and VCAM-1 (Pober, J. S., et al., J. Immunol., 1993, 150, 5114-5123).
Antisense oligonucleotides to c-raf, Ha-ras and JNK2 are known, but have not previously been shown to inhibit cell adhesion molecule expression. The relationship between these TNF-.alpha. signaling molecules and cell adhesion molecule expression has not been fully delineated. c-raf antisense oligonucleotides are disclosed in U.S. Pat. Nos. 5,563,255 and 5,656,612, herein incorporated by reference. Ha-ras antisense oligonucleotide are disclosed in U.S. Pat. Nos. 5,576,208 and 5,582,986, herein incorporated by reference. JNK2 antisense oligonucleotides are disclosed by Bost, F., et al. (J. Biol. Chem. 1997, 272, 33422-33429). Inhibitors of the TNF-.alpha. signaling molecules, c-raf, Ha-ras and JNK2 have not been used to modulate expression of cell adhesion molecules and represent a novel approach.